The invention relates to an active substance and composition inhibiting tumorous proliferation in ectoderm derived tissues and to the process for producing thereof.
One of the most serious illness in the 20th century is cancer. Many experimental works have the aim to discover the reason of the formation of tumorous tissue proliferation.
The formation of tumorous cells and the properties of these cells are discussed in the following publications: Cancer Res. 47., p. 1473, 1987; European Journal of Cancer 15., p. 585, 1979; Science 197., p. 893, 1977; Int. Rev. Exp. Pathol, 16., p. 207, 1976.
Several drugs and compositions are known which inhibit the tumorous proliferation. The active ingredient of the composition is either natural or synthetic. Thus, for example the tumor inhibiting effect of natural active substances isolated from plants is discussed in the following papers: Ukrain. J. Cell. Biochem., 1987., Suppl. 11A: 53; J. Tumor Marker Oncol., 1988, 3., p. 463-465.
In the course of our research work we suggested that the reason of the cell proliferation in ectoderm derived differentiated tissues could be that because of the liberation of the previously suppressed primordial reproduction code the new cells start an endless reproduction. The multiplying new cells form an aggregate (tumor tissue) and by means of their metalloprotease enzyme they lyse the surrounding tissues. Growing in such a diffuse way they advance into their environment, and after causing erosion of vessels they get into the lymphatic system and blood circulation. The detached cells can induce neoplasm in places far from the primary tumor. This cell proliferation schema corresponds to the cell reproduction according to the endless cell reproduction code in the early stage of phylogenesis.
In primordial times to the formation of an organic structure instead of a cell aggregate the suppression of the endless cell reproduction code was necessary and the building of a code into the cell which would ensure the formation and the function of the given organization.
It is known that in animals including humanoids, the DNA contains the code system of formation and function of organizations.
In the coarse of our studies we have discovered that the basic event which causes the malignous transformation of the cell functioning previously differentiatedly, takes place on DNA level.
Our aim was to provide an active substance containing those codes which are capable to inhibit the endless and speedy cell reproduction. We have found that the substance containing this genetic code, probably connected to RNA, can be isolated from fertilized ovum.
It is known from WO-A-96/39428, WO-A-91/07435, WO-A-89/09606, that it is possible to produce proteins from Rana pipiens fertilized eggs which have antitumor effect. This active substance has a different composition, different molecular weight (preferable 4000-6000 dalton) and effect, because of the different starting material.
For the preparation of the active substance according to the invention human or animal derived fertilized ovum, preferably the fertilized ovum of a fish is used as starting material. The fertilized ovum of the following fish species can advantageously be applied: Cyprinus carpio, Tinca tinca, Gobio gobio, Gobio uranoscopus, Barbus barbus, Barbus meridionalis petenyii, Hypophtalmichthys molitrix, Aristichtys nobilis, Carassius carassius, Carassius auratus gibelio, Leuciscus sonffia agassizi, Chondrostoma nasus, Pseudorasbora parva, Leuciscus idus, Cienopharyngodon idella, Leuciscus cephalus, Scardinius erythrophthalmus, Leuciscus leuciscus, Leuciscus virgo, Rutilus rutilus, Phoxinus phoxinus, Phoxinus percnurus, Leucaspius delineatus, Rhodeus sericeus amarus, Pelecus cultratus, Aspius aspius, Alburnus mento, Alburnus alburnus, Alburnoides bipunciatus, Vimba vimba, Blicca bjoerkna, Abramis brama, Abramis ballerus, Abramis sapa, Carassius auratus. 
In case when the fertilized ovum of a sea water fish or fresh water fish is used as starting material the fertilization is performed artificially and the embryonic development is interrupted at a stage where the endless reproduction stops and differentiated development starts. Therefore the fertilization is performed for 24 hours at most, preferably for 14-16 hours. The fertilized ovum prepared in this way is worked up according to the invention. The fertilization is performed preferable at 12-14xc2x0 C. Thus, the subject of the present invention is an active substance inhibiting tumorous proliferation appearing in ectoderm derived tissueswhich is derivedfrom fertilezed ovum of human beings or sea water fishes or fresh water fishes, as well as a composition containing such substance. The subject of the invention the process for producing the active substance. The process is comprising the following steps: the fertilized ovum of human beings or sea water fishes or fresh water fishes is subjected to cell wall destruction with stirring 24 hours at most, preferably 14-16 hours after fertilization, then the mixture is ultracentrifuged, the supernatant is separated, the sediment is homogenized and ultracentrifuged several times in the coarse of which the supernatant and the sediment are separated, then all the supernatants are combined and centrifuged preferably at 8,000-15,000 rpm, the supernatant obtained is separated and diluted with physiological saline solution, preferably in 1:2 ratio by weight, the mixture is filtered at least twice through gel filters, then the filtered liquid is lyophilized. The subject of the invention is also the process for producing the composition which is containing the active substance. According to this process the active substance is mixed with additives or optionally is diluted with a diluent, preferably with physiological saline solution or dextrose solution or sterile distilled water, preferably in 1:2 ratio by weight. The fertilized ovum subjected to cell wall destruction can be stored in a deep-freezer preferably at xe2x88x9270xc2x0 C. until final workup. Before use the frozen material is allowed to thaw in a water bath of 20-25xc2x0 C. for 2-3 hours. The fertilized ovum subjected to cell wall destruction is ultracentrifuged at 25,000-30,000 rpm. Then the supernatant containing RNA is separated from the sediment consisting of mainly high molecular weight proteins and carbohydrates.
The sediment is ultracentrifuged again to separate the supernatant more properly from the fraction consisting of high molecular weight protein and carbohydrates. The combined supernatant containing the active substance is repeatedly centrifuged in order to effect a proper separation, then the supernatant obtained in this way is subjected to gel filtration for several times to remove proteins and bacteria.
The active substance prepared can be lyophilized or mixed with physiological solutions to form composition. In the composition the ratio of the active substance and the physiological solution can be optional, but preferably we prepare solutions in which the ratio of the active substance and the additives is 1:2 by weight.
The lyophilized active substance can be stored for unlimited time, the composition prepared by dilution with physiological saline solution or dextrose solution can be stored at xe2x88x9215xc2x0 C. for 4 years.
We presume that with the supernatant of the fertilized ovum we separate the RNA from the carbohydrates and the high molecular weight proteins.
As tumor inhibiting agent the composition is to be administered intravenously in form of a cure. To a human patient of 70 kg body weight 1.5 ml of active substance is administered after dilution to 10 ml of volume, preferably with physiologic saline solution.
The treatment is performed according the so called Besredka method. Before starting the cure 0.1 ml solution containing the active substance is diluted to 10 ml with physiological saline solution and administered to the patient. If sensitivity is experienced, the so called anaphylactic shock develops, the active substance content is increased gradually to 1.5 ml volume. In our experiments we have not observed such a sensitivity.
One cure lasts for 14 days, and every day 1.5 ml of active substance diluted to 10 ml with physiological saline solution (counted for 70 kg of body weight) is administered intravenously. After that, for a further 14 days the same composition is administered every other day.
After half a year a control is performed and if the tumor inhibiting effect was not satisfactory, an additional 14 days cure is applied with daily intravenous administration.